1. Field of the Invention
This invention relates to specimen holders for maintaining a specimen at a low temperature during visualization, imaging or analysis. More specifically it relates to holders utilized for transmission electron microscopy (TEM) and particularly to the Dewar used for the containment of liquid nitrogen and the subsequent cooling of the TEM sample placed on a retractable cartridge and positioned within a cryogenically cooled shield.
2. Description of the Prior Art
There are a variety of imaging technologies which have developed to observe and analyze specimens at the molecular and/or atomic level. These include optical, electron, x-ray and photon microscopy together with associated imaging and analysis. Cryo electron microscopy, or Cryo EM, is a powerful technique for studying frozen hydrated biological specimens in transmission electron microscopy. To generate results with minimum artifacts, specimens are rapidly frozen and then imaged in a fully hydrated state. This reduces the detrimental effects of fixatives or stains that are commonly used to prepare microscopy specimens at more ambient temperatures. Cryo EM is extremely beneficial for studying proteins, viruses, macromolecular assemblies, vesicles/liposomes, organelles, and cells in more native conditions. In order to obtain a TEM image the specimen needs to be sufficiently thin to allow for the transmission of electrons therethrough. As with all conventional TEM imaging, the TEM image is then formed by electron interactions with the specimen. The quality and usability of TEM images increases with improved resolution. Biological specimen quality is highly dependent on the method of preparation. Typical preparation includes rapidly vitrifying a thin film of suspension by freezing it in an extremely cold material, such as liquid ethane. The specimen is ultimately transferred to the TEM while being maintained in the frozen state and at a consistently low temperature and examined in its fully hydrated state. Alternatively, a bulk specimen can be cryoprotected, high pressure frozen, cryosectioned and transferred to the imaging or analytical device. The mounting/support and transfer of specimen to a TEM, as an illustrative example, for subsequent imaging has typically been performed with the help of a cryotransfer specimen holder. These prior art TEM specimen holders, consistent with TEM specimen holders, generally, comprise longitudinal rods of a given length to support and mount the specimen near one end of the rod. The rod end of the TEM specimen holder is inserted into the microscope and placed between the components of the electron optics. As is well known to those skilled in the art, these components, by physical necessity, allow for only a very small and dimensionally constrained specimen or support contained thereon. With respect to biological specimens, as indicated above, the specimen must also be maintained at a low temperature, preferably below −140° C. during the transfer, imaging and analysis process and while located within the constrained space of the microscope. This is because it is desirable to maintain the ice component of the specimen in an amorphous state which exists below −140° C. Above −140° C., the ice will adopt a crystalline form which is detrimental to imaging and analysis. During transfer and while the specimen holder is not inserted into the imaging or analysis device, it is also essential to physically protect the specimen, as it is highly vulnerable to physical and environmental damage and/or contamination from water vapor and other sources.
The physical port of a microscope, for example, which accepts and restrains the specimen holder is known as the goniometer. It is a micromanipulator for moving the specimen holder, and thus the specimen itself, in the X, Y, Z, α and β tilt directions. This helps to focus the electron beam onto the desired region of the specimen and at the precise angle/orientation necessary to observe the relevant characteristics of the specimen. As stated, the goniometer is used to tilt the specimen holder inside the column of the microscope relative to the electron imaging beam. Angular displacement of the specimen while mounted within the microscope is an extremely important feature for cryotomography in order to obtain three-dimensional, or 3D, information for life science applications. This same methodology is applicable to the physical sciences. To generate 3D information, the sample is imaged at various tilt angles and/or orientations. The two-dimensional projections are then recombined to produce a composite 3D image.
While mounted on the specimen holder, the specimen is maintained at the required low temperature through the use of a cooling medium which reduces the temperature of portions of the specimen holder and the specimen itself. This cooling medium, typically liquid nitrogen, is stored in an insulated container mounted to one end of the specimen holder, typically identified as a Dewar. The Dewar is a component of the specimen holder and it comprises a highly reflective inner vessel enclosed within an evacuated housing. The vacuum within the evacuated housing, coupled with the materials utilized for the construction of the device, thermally isolates the inner vessel from the housing. A rod or other thermal conductor assembly provides the thermal contact between the specimen and the receptacle for the cooling medium present in the Dewar. The conductor is typically constructed of a material having high thermal conductivity such as silver or copper. The cooling medium is utilized to remove the heat from the specimen support and specimen to maintain the same at the necessary low temperature.
Modern day transmission electron microscopes are capable of achieving atomic scale resolution. However, image quality and resolution are highly dependent upon reduction of specimen displacement through vibrations and drift induced from the holder during imaging or analysis. In practice, these environmental and other conditions need to be minimized to achieve optimum resolution. At atmospheric pressure, liquid nitrogen boils at −196° C. In many prior art specimen holder designs, a solid conductor rod within the holder is in contact with the Dewar and the specimen support in the form of a receptacle tip. As the liquid nitrogen or other cooling medium boils off under ambient atmospheric conditions, vibrations are formed by the turbulence in the medium. The conductor rod transmits these vibrations directly to the specimen tip, causing the specimen to vibrate during imaging and analysis. The rigid contact between the Dewar and the cooling assembly of the prior art devices further introduces physical stresses on the device during thermal expansion and contraction.
Most Dewar devices are open to ambient atmosphere to permit the boil off of the liquid medium and to minimize the retention of expanding warm gas medium, which has deleterious effects relating to pressure within the vessel. The devices are rigidly constructed such that any displacement of the specimen holder results in a corresponding displacement of the specimen. In tomography, higher tilt angles of the specimen during imaging yield more accurate and detailed 3D reconstructions. The ability to increase the tilt of the specimen holder is limited, however, by the possibility of spillover of the liquid medium from the Dewar, as well as a thermal gradient induced into the walls of the container, which result in unsatisfactory results, including vibrations, drift and, potentially, spilled liquid nitrogen in the laboratory. Avoidance of this condition substantially limits the ability to tilt the specimen.
Another shortcoming of prior art cryogenic specimen holder designs is the ability to constrain thermal distortions of the device itself. Thermal variations lead to the expansion or contraction of materials. The thermal gradient present between the Dewar and the specimen cartridge, as a function of distance and time, as well as changing environmental conditions along the length of the holder, causes unpredictable and dynamic dimensional changes, resulting in specimen drift from the nominal position within the imaging device. It is desirable, therefore, to maintain the assembly at a constant, low steady state temperature with a minimum thermal gradient. Moreover, any thermal contact between the conductor assembly, which extends within, but dimensionally separate from the outer holder barrel, may introduce additional heat or, at a minimum, temperature variations within the system. In addition, since the exterior holder barrel is in contact with the microscope goniometer during imaging, any such contact between the conductor assembly and the outer holder barrel leads to an undesirable thermal path from the microscope, a large warm heat sink, and the specimen, causing additional drift.
During imaging and analysis of the specimen, constant evaporation of the cooling medium also results in a drop in the volume of liquid present in the Dewar. This necessitates the physical interface between the cooling assembly and the Dewar to be located at a point most likely to be in direct contact with the cooling medium, which is the bottom of the Dewar. At any given time, a temperature gradient exists along the wall of the Dewar, being coolest at the points of contact directly adjacent the cooling medium and increasing in temperature with increasing distance from the surface of the cooling medium. Loss of direct contact with the cooling medium immediately adjacent the interface between the cooling assembly and the Dewar causes the temperature to rise in the assembly and further exacerbates drift of the specimen during imaging.
Cryotransfer holders like those described in Swann et al., U.S. Pat. No. 5,753,924, have been developed to maintain samples at the desired temperature and to prevent frost from forming on the specimen during transfer. As illustrated in FIG. 1 of Swann, the holder 10 includes a holder body 12 and a specimen tip 14 with a source of cooling for the tip. The specimen tip 14 includes a support grid 16 of thermally conductive material and a tab portion 24 that is adapted to be secured to the specimen tip (see FIG. 5). A cryoshield is formed by an opening in the specimen holder tip 14. To load the specimen grid 16 into the tip 14 of holder body 12, tab 24 is inserted through a slot 40 in specimen tip holder 14 (see FIG. 4) which forms a cryoshield for the specimen. The specimen grid 16 is moved from an extended position to a retracted position by a drawbar 46, which is in thermal contact with support grid 16 and extends along the longitudinal axis of holder body 12.
The cryotransfer holder of Swann suffers from several disadvantages. In this design, the drawbar is in rigid thermal contact with the support grid and the holder specimen tip, thus acting as a potential source of heat load. This requires much greater energy extraction to cool the entire assembly to the desired temperature. Extraneous heat may cause thermal expansion and contraction of the cooling rod and drawbar. The rigid contact directly transfers all such movement to the specimen resulting in a loss of image resolution because of drift and vibrations. Finally, the proprietary design of the specimen grid, which requires insertion of a tab to secure the grid into the cryoholder, makes the system incompatible with any independent, standard transmission electron microscope specimen disks, which are 3 mm in diameter, or any other shapes now in use or in development.
The Dewar assembly of the type described by Gallagher et al., U.S. Pat. No. 5,302,831 has been developed in an attempt to maintain constant contact between the liquid nitrogen supply and the cold finger assembly. FIG. 2 of Gallagher illustrates a trapezoidal or truncated triangular shaped Dewar 50 with a cold finger assembly 14. A copper braided strap 122 is secured on one end through a lug 118 to the bottom wall 94 of liquid nitrogen vessel. The other end of the copper braided strap 122 is secured through a lug to a portion of the cold finger assembly 114. The triangular Dewar 50 is tapered at the bottom so that liquid nitrogen always fills the front portion of vessel 90 adjacent to the intersection of the front wall 98 and the bottom wall 94. Dewar 50 is angled so that the liquid nitrogen is forced into a portion of Dewar 50 that is in constant contact with the cold finger assembly 114 and strap 122 at a fixed angle in the range of 0° to 60°. The vessel 90 top wall 102 has an opening 103 for the addition of liquid nitrogen to Dewar 50.
The Dewar described in Gallagher is used to cool a radiation detector cold finger, which has different physical constraints than a TEM specimen holder, including, most importantly, that a specimen is not cooled thereby, merely a detector. This Dewar is not built to satisfy any of the specimen vibration and drift requirements necessary for proper TEM imaging. Also, cooling a radiation detector does not require or even permit the Dewar to be tilted about the longitudinal axis of the cold finger. Gallagher's trapezoidally shaped Dewar will not contain the liquid nitrogen at higher tilt angles, causing the liquid nitrogen to boil and spill over leading to substantial sample drift. The three corners present near the Dewar neck will also trap nitrogen gas, causing vibrations, which in turn would also limit image resolution.
There remains a need, therefore, for a cryogenic holder adapted to be rotated about the longitudinal axis of the holder, which will help tilt the specimen within the microscope while maintaining optimum thermal transmission between the liquid cooling medium and the specimen at all times. The specimen support cartridge has to be vibrationally isolated from the cooling medium and the exterior sections of the holder have to be thermally isolated from the cooling medium to provide additional reduction of sample drift and vibration and to reduce thermal stresses within the device. In addition, there is a need for a holder that includes a cartridge specimen tip that receives a standard sized specimen and optionally and controllably shields the specimen during transport to prevent contamination.